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Food/farmed animals
Characterisation of InlA truncation in Listeria monocytogenes isolates from farm animals and human cases in the province of Quebec Philippe Fravalo,1 Tamazight Cherifi,1 Kersti Dina Neira Feliciano,1 Ann Letellier,1 Julie-Hélène Fairbrother,2 Sadjia Bekal3
To cite: Fravalo P, Cherifi T, Neira Feliciano KD, et al. Characterisation of InlA truncation in Listeria monocytogenes isolates from farm animals and human cases in the province of Quebec. Veterinary Record Open 2017;4:e000199. doi:10.1136/ vetreco-2016-000199 Received 2 August 2016 Revised 21 December 2016 Accepted 21 February 2017
1 University of Montreal, Faculty veterinary medicine, Dpt Pathology Microbiology 3190 Sicotte J2S2M2, Saint Hyacinthe, QC, Canada 2 Laboratoire d’épidémiosurveillance animale du Québec Ministry of Agriculture, Fisheries and Food, Saint Hyacinthe, QC, Canada 3 Provincial Public Health Laboratory (LSPQ), QC, Canada
Correspondence to Dr Philippe Fravalo, CRSV, Pathology-Microbiology, faculty of veterinary medicine, University of Montreal, 3190 Sicotte street, Saint-Hyacinthe, QCJ2S2M2, Canada; philippe. fravalo@umontreal.ca
ABSTRACT
The introduction of Listeria monocytogenes into the food production chain is a concern, with numerous grouped cases of listeriosis associated with milk-derived or porkderived products have been documented. Management of this zoonotic pathogen considers all strains as an equal risk. Recently, a new perspective for characterisation of strain virulence was introduced with the discovery of the unaltered sequence of InlA as a determinant of strain virulence; this has also been reported as an infrequent finding among so-called environmental strains, that is, strains isolated from food or from surfaces in food industries. The aim of this study was to differentiate L monocytogenes strains isolated from animal cases versus those from human cases and to differentiate clinical strains from environmental ones using a Caenorhabditis elegans virulence testing model. In Quebec in 2013/2014, the surveillance of L monocytogenes clinical isolates registered a total of 20 strains of animal origin and 16 pulsed-field gel electrophoresis types isolated from human cases. The mixed PCR multiplex agglutination protocol used for geno-serotyping clearly discriminated genogroup IVB strains from bovine and human origins. The presence of a premature stop codon single nucleotide polymorphism in the inlA gene sequence in clinical strains and the identical behaviour of particular strains in the C elegans model are discussed in this paper from the perspective of industrial management of L monocytogenes risk.
INTRODUCTION Listeria monocytogenes is a great concern both for industrial and public health stakeholders. These bacteria cause listeriosis, a foodborne disease acquired through the consumption of contaminated ready-to-eat products, including milk or meat products. Pork meat has been associated with largescale outbreaks in the past (de valk and others 2001) and is considered a risk factor, particularly for sensitive populations such as pregnant women, children and the elderly. In the last few years, the increase in human cases (not associated with intensified surveillance), mainly for the elderly, has increased
the concern surrounding management of L monocytogenes in food in Canada and Europe (Lomonaco and others 2015). One critical question to answer is whether the virulence of L monocytogenes has increased. The determinants of virulence have been studied, and there is a large amount of data that allow for discrimination of strains from lineages III or IV from lineages I and II (Ragon and others 2008). Lineages I and II are considered to be the most worrisome because of their potential to cause outbreaks or sporadic listeriosis. Among such strains, discrimination based on pathogenic properties still represents a challenge. A few years ago, the very promising concept of virulence determination through inlA gene sequencing was introduced (Van Stelten and others 2010, Van Stelten and others 2011). Published studies revealed the presence of a premature stop codon for internalin A translation. Because this stop codon inactivated this determinant of the first step of pathogenicity in the digestive tract, a key determinant for virulence differentiation of strains appeared to have been discovered. In Quebec, human listeriosis surveillance works hand-in-hand with food surveillance. Few strains isolated from clinical cases in farms (animal primary productions) are available, so we took the opportunity that arose to test virulent strains isolated from animals or humans with clinical signs of listeriosis. After we performed the serovar determination and comparison according to strain origin, we wanted to confirm if the inlA sequences were complete in these strains and to test their behaviour in a Caenorhabditis elegans infection model, a very attractive but controversial model of pathogenicity testing for L monocytogenes (Thomsen and others 2006, Karthikeyan and others 2015) (see discussion). No relationship between
Fravalo P, et al. Vet Rec Open 2017;4:e000199. doi:10.1136/vetreco-2016-000199
1
Downloaded from http://vetrecordopen.bmj.com/ on September 18, 2017 - Published by group.bmj.com
Open Access InlA integrity and behaviour of L monocytogenes strain in C elegans has actually been described. Some strains isolated during a previous study involving pig primary production (Larivière-Gauthier and others 2014) were added to the study’s strain collection because only some of them showed stop codon in their inlA sequences and because these strains were (based on pulsotype comparison) frequently or not associated with clinical human cases. These additional strains were expected to show opposite virulence potentials. The aim of this study was to analyse inlA sequences from strains isolated from clinical forms of human or animal listerioses in Quebec and to compare the behaviour of some selected strains in a C elegans virulence model. MATERIALS AND METHODS Strain collections Strains were collected from L monocytogenes surveillance in 2013/2014: 20 strains were obtained from animal disease surveillance (laboratory of the Ministry of Agriculture, Fisheries and Food, Quebec (MAPAQ)), among which 13 were included in this study because they satisfied the required ‘clinical’ criteria (ie, isolated from organs after animal necropsy, excluding isolation from milk and silage) and 16 strains isolated from clinical cases in humans were provided by the Provincial Public Health Laboratory (public health surveillance). The collection was completed with four strains of pig origin isolated in a previous project (Larivière-Gauthier and others 2014). These strains showed a single nucleotide polymorphism (SNP) premature stop codon in the inlA sequence (Pulso1 and Pulso9 strains) or an integrated inlA sequence (Pulso4 and Pulso6 strains). Strains Pulso6 and Pulso9 showed 100% similarity between pulsed-field gel electrophoresis (PFGE) banding patterns with strains recovered from clinical human cases; strains Pulso1 and Pulso4 have not previously been identified in clinical human surveillance. These added strains were expected to show opposite virulence potentials. Serotyping L monocytogenes strains were serotyped based on a combination of multiplex PCR-based geno-serogrouping, completed by detection of flaA (Kérouanton and others 2010) and agglutination against discriminatory O serum (OI, OVII, OVIII; Oxoid Thermo Fisher Scientific, Nepean, ON, Canada) (Burall and others 2011). InlA sequencing The inlA gene sequences were determined after overlapping amplification (Ragon and others 2008) using the Sanger method at the Centre d’Innovation Génome Québec (Applied Biosystems 3730xl DNA analyzer). InlA gene sequencing was extensively analysed, and the occurrence and position of SNP before in silico translation of the sequences were compared. Sequences were aligned and screened for mutations causing a premature stop codon or amino acid deletion using ClustalX 2.1 software, 2
with inlA L monocytogenes EGD-e (NCBI: NC_003210.1) as reference. C elegans virulence model Bacterial strains (Fig 1) were cultured 24 hours at 37°C in BHI (Brain Heart Infusion), acid shocked (0.1 N HCl pH 4.5, one hour) then washed and suspended in phosphate-buffered saline (PBS) (concentration, see Fig 1) before being used in trials. Strain N2 worms, maintained for five days on nematode-growing medium plates and inoculated with Escherichia coli OP50 feeding strain at 25°C, were harvested (non-synchronised culture), washed three times in PBS, then inoculated in 96-well plates in PBS and maintained for five more days at 30°C with daily addition of fresh bacteria. Observation of worm viability (based on worm locomotion and/or pharyngeal pumping and assessed under an inverted phase contrast microscope according to Thomsen and others (2006) allowed determination of a living worm count of 15 mature worms and 30 L1/L2 larvae in each of the tested conditions. The distribution of number of living worms counts (n=8) were compared with control conditions (Mann-Whitney U test; SPSS Statistics V.17, Licenced U de Montreal, α=0.05) RESULTS All 16 of the strains of human origin were considered clinical because they were isolated from hospitalised patients. From the 20 strains provided by MAPAQ, only 13 strains strictly associated with clinical listeriosis in animals were included. It should be noted that we excluded strains isolated from milk tanks and silage, all belonging to the 1/2a serovar. Although serotyping revealed that the greater proportion of clinical strains (regardless of origin) belonged to the serovar 1/2a (18/29, 62%), there were still some strains (nine out of 29 with similar proportions from humans and animals, that is, 5/16 and 4/13, respectively) that belonged to the IVB serogroup (a serogroup that contains 4b and 4a/ab serovars). Focusing on this IVB group, a clear distinction appeared where only 4b serovar strains were found in human surveillance, whereas strains shown in ruminant clinical cases all belonged to 4a/4ab serovars (table presented in Fig 2). No SNP occurred on the sequence coding for the InlA LPXTG domain. Moreover, inlA sequencing showed that a majority of strains had a complete sequence (table presented in Fig 2). But it should be noted that two out of the 16 strains isolated from clinical listeriosis cases had a premature stop codon; the mutations were expected to induce truncations of InlA at 700 and 762 amino acid (aa) positions, respectively. One of these—which showed the longest InlA size but lost LPXTG domain—was used in the C elegans virulence model, as well as another strain presenting a complete sequence (tables presented in Figs 1,2). Four more strains (different PFGE type) from porcine origin were added; some had been recurrently detected in production (and food) but had never been Fravalo P, et al. Vet Rec Open 2017;4:e000199. doi:10.1136/vetreco-2016-000199
Downloaded from http://vetrecordopen.bmj.com/ on September 18, 2017 - Published by group.bmj.com
Open Access
FIG 1: Listeria monocytogenes virulence on Caenorhabditis elegans model isolated in human (h) or pig (s). h s: strains from animal origin related to human cases (identical pulsed-field gel electrophoresis profile). Comp: complete InlA; PSC 700: truncated InlA due to the presence of a 700 aa position premature stop codon. Comp.#: complete but with substitution of first amino acids. aa, amino acid.
associated with human cases (table presented in Fig 1; Pulso1 and Pulso4 strains). The two others were kept because of their 100% homology with strains involved in clinical human cases (sporadically for strain Pulso9 or recurrently for strain Pulso6; table presented in Fig 1). After five days, unfed C elegans worms were almost all dead. Using E coli OP 50 as a control, viability was maintained at 74% after five days, both in larvae and in mature worm forms. By comparison, L monocytogenes lowered the viability of the worms and significantly so for the larvae forms (Mann-Whitney U test, P
Food/farmed animals
Characterisation of InlA truncation in Listeria monocytogenes isolates from farm animals and human cases in the province of Quebec Philippe Fravalo,1 Tamazight Cherifi,1 Kersti Dina Neira Feliciano,1 Ann Letellier,1 Julie-Hélène Fairbrother,2 Sadjia Bekal3
To cite: Fravalo P, Cherifi T, Neira Feliciano KD, et al. Characterisation of InlA truncation in Listeria monocytogenes isolates from farm animals and human cases in the province of Quebec. Veterinary Record Open 2017;4:e000199. doi:10.1136/ vetreco-2016-000199 Received 2 August 2016 Revised 21 December 2016 Accepted 21 February 2017
1 University of Montreal, Faculty veterinary medicine, Dpt Pathology Microbiology 3190 Sicotte J2S2M2, Saint Hyacinthe, QC, Canada 2 Laboratoire d’épidémiosurveillance animale du Québec Ministry of Agriculture, Fisheries and Food, Saint Hyacinthe, QC, Canada 3 Provincial Public Health Laboratory (LSPQ), QC, Canada
Correspondence to Dr Philippe Fravalo, CRSV, Pathology-Microbiology, faculty of veterinary medicine, University of Montreal, 3190 Sicotte street, Saint-Hyacinthe, QCJ2S2M2, Canada; philippe. fravalo@umontreal.ca
ABSTRACT
The introduction of Listeria monocytogenes into the food production chain is a concern, with numerous grouped cases of listeriosis associated with milk-derived or porkderived products have been documented. Management of this zoonotic pathogen considers all strains as an equal risk. Recently, a new perspective for characterisation of strain virulence was introduced with the discovery of the unaltered sequence of InlA as a determinant of strain virulence; this has also been reported as an infrequent finding among so-called environmental strains, that is, strains isolated from food or from surfaces in food industries. The aim of this study was to differentiate L monocytogenes strains isolated from animal cases versus those from human cases and to differentiate clinical strains from environmental ones using a Caenorhabditis elegans virulence testing model. In Quebec in 2013/2014, the surveillance of L monocytogenes clinical isolates registered a total of 20 strains of animal origin and 16 pulsed-field gel electrophoresis types isolated from human cases. The mixed PCR multiplex agglutination protocol used for geno-serotyping clearly discriminated genogroup IVB strains from bovine and human origins. The presence of a premature stop codon single nucleotide polymorphism in the inlA gene sequence in clinical strains and the identical behaviour of particular strains in the C elegans model are discussed in this paper from the perspective of industrial management of L monocytogenes risk.
INTRODUCTION Listeria monocytogenes is a great concern both for industrial and public health stakeholders. These bacteria cause listeriosis, a foodborne disease acquired through the consumption of contaminated ready-to-eat products, including milk or meat products. Pork meat has been associated with largescale outbreaks in the past (de valk and others 2001) and is considered a risk factor, particularly for sensitive populations such as pregnant women, children and the elderly. In the last few years, the increase in human cases (not associated with intensified surveillance), mainly for the elderly, has increased
the concern surrounding management of L monocytogenes in food in Canada and Europe (Lomonaco and others 2015). One critical question to answer is whether the virulence of L monocytogenes has increased. The determinants of virulence have been studied, and there is a large amount of data that allow for discrimination of strains from lineages III or IV from lineages I and II (Ragon and others 2008). Lineages I and II are considered to be the most worrisome because of their potential to cause outbreaks or sporadic listeriosis. Among such strains, discrimination based on pathogenic properties still represents a challenge. A few years ago, the very promising concept of virulence determination through inlA gene sequencing was introduced (Van Stelten and others 2010, Van Stelten and others 2011). Published studies revealed the presence of a premature stop codon for internalin A translation. Because this stop codon inactivated this determinant of the first step of pathogenicity in the digestive tract, a key determinant for virulence differentiation of strains appeared to have been discovered. In Quebec, human listeriosis surveillance works hand-in-hand with food surveillance. Few strains isolated from clinical cases in farms (animal primary productions) are available, so we took the opportunity that arose to test virulent strains isolated from animals or humans with clinical signs of listeriosis. After we performed the serovar determination and comparison according to strain origin, we wanted to confirm if the inlA sequences were complete in these strains and to test their behaviour in a Caenorhabditis elegans infection model, a very attractive but controversial model of pathogenicity testing for L monocytogenes (Thomsen and others 2006, Karthikeyan and others 2015) (see discussion). No relationship between
Fravalo P, et al. Vet Rec Open 2017;4:e000199. doi:10.1136/vetreco-2016-000199
1
Downloaded from http://vetrecordopen.bmj.com/ on September 18, 2017 - Published by group.bmj.com
Open Access InlA integrity and behaviour of L monocytogenes strain in C elegans has actually been described. Some strains isolated during a previous study involving pig primary production (Larivière-Gauthier and others 2014) were added to the study’s strain collection because only some of them showed stop codon in their inlA sequences and because these strains were (based on pulsotype comparison) frequently or not associated with clinical human cases. These additional strains were expected to show opposite virulence potentials. The aim of this study was to analyse inlA sequences from strains isolated from clinical forms of human or animal listerioses in Quebec and to compare the behaviour of some selected strains in a C elegans virulence model. MATERIALS AND METHODS Strain collections Strains were collected from L monocytogenes surveillance in 2013/2014: 20 strains were obtained from animal disease surveillance (laboratory of the Ministry of Agriculture, Fisheries and Food, Quebec (MAPAQ)), among which 13 were included in this study because they satisfied the required ‘clinical’ criteria (ie, isolated from organs after animal necropsy, excluding isolation from milk and silage) and 16 strains isolated from clinical cases in humans were provided by the Provincial Public Health Laboratory (public health surveillance). The collection was completed with four strains of pig origin isolated in a previous project (Larivière-Gauthier and others 2014). These strains showed a single nucleotide polymorphism (SNP) premature stop codon in the inlA sequence (Pulso1 and Pulso9 strains) or an integrated inlA sequence (Pulso4 and Pulso6 strains). Strains Pulso6 and Pulso9 showed 100% similarity between pulsed-field gel electrophoresis (PFGE) banding patterns with strains recovered from clinical human cases; strains Pulso1 and Pulso4 have not previously been identified in clinical human surveillance. These added strains were expected to show opposite virulence potentials. Serotyping L monocytogenes strains were serotyped based on a combination of multiplex PCR-based geno-serogrouping, completed by detection of flaA (Kérouanton and others 2010) and agglutination against discriminatory O serum (OI, OVII, OVIII; Oxoid Thermo Fisher Scientific, Nepean, ON, Canada) (Burall and others 2011). InlA sequencing The inlA gene sequences were determined after overlapping amplification (Ragon and others 2008) using the Sanger method at the Centre d’Innovation Génome Québec (Applied Biosystems 3730xl DNA analyzer). InlA gene sequencing was extensively analysed, and the occurrence and position of SNP before in silico translation of the sequences were compared. Sequences were aligned and screened for mutations causing a premature stop codon or amino acid deletion using ClustalX 2.1 software, 2
with inlA L monocytogenes EGD-e (NCBI: NC_003210.1) as reference. C elegans virulence model Bacterial strains (Fig 1) were cultured 24 hours at 37°C in BHI (Brain Heart Infusion), acid shocked (0.1 N HCl pH 4.5, one hour) then washed and suspended in phosphate-buffered saline (PBS) (concentration, see Fig 1) before being used in trials. Strain N2 worms, maintained for five days on nematode-growing medium plates and inoculated with Escherichia coli OP50 feeding strain at 25°C, were harvested (non-synchronised culture), washed three times in PBS, then inoculated in 96-well plates in PBS and maintained for five more days at 30°C with daily addition of fresh bacteria. Observation of worm viability (based on worm locomotion and/or pharyngeal pumping and assessed under an inverted phase contrast microscope according to Thomsen and others (2006) allowed determination of a living worm count of 15 mature worms and 30 L1/L2 larvae in each of the tested conditions. The distribution of number of living worms counts (n=8) were compared with control conditions (Mann-Whitney U test; SPSS Statistics V.17, Licenced U de Montreal, α=0.05) RESULTS All 16 of the strains of human origin were considered clinical because they were isolated from hospitalised patients. From the 20 strains provided by MAPAQ, only 13 strains strictly associated with clinical listeriosis in animals were included. It should be noted that we excluded strains isolated from milk tanks and silage, all belonging to the 1/2a serovar. Although serotyping revealed that the greater proportion of clinical strains (regardless of origin) belonged to the serovar 1/2a (18/29, 62%), there were still some strains (nine out of 29 with similar proportions from humans and animals, that is, 5/16 and 4/13, respectively) that belonged to the IVB serogroup (a serogroup that contains 4b and 4a/ab serovars). Focusing on this IVB group, a clear distinction appeared where only 4b serovar strains were found in human surveillance, whereas strains shown in ruminant clinical cases all belonged to 4a/4ab serovars (table presented in Fig 2). No SNP occurred on the sequence coding for the InlA LPXTG domain. Moreover, inlA sequencing showed that a majority of strains had a complete sequence (table presented in Fig 2). But it should be noted that two out of the 16 strains isolated from clinical listeriosis cases had a premature stop codon; the mutations were expected to induce truncations of InlA at 700 and 762 amino acid (aa) positions, respectively. One of these—which showed the longest InlA size but lost LPXTG domain—was used in the C elegans virulence model, as well as another strain presenting a complete sequence (tables presented in Figs 1,2). Four more strains (different PFGE type) from porcine origin were added; some had been recurrently detected in production (and food) but had never been Fravalo P, et al. Vet Rec Open 2017;4:e000199. doi:10.1136/vetreco-2016-000199
Downloaded from http://vetrecordopen.bmj.com/ on September 18, 2017 - Published by group.bmj.com
Open Access
FIG 1: Listeria monocytogenes virulence on Caenorhabditis elegans model isolated in human (h) or pig (s). h s: strains from animal origin related to human cases (identical pulsed-field gel electrophoresis profile). Comp: complete InlA; PSC 700: truncated InlA due to the presence of a 700 aa position premature stop codon. Comp.#: complete but with substitution of first amino acids. aa, amino acid.
associated with human cases (table presented in Fig 1; Pulso1 and Pulso4 strains). The two others were kept because of their 100% homology with strains involved in clinical human cases (sporadically for strain Pulso9 or recurrently for strain Pulso6; table presented in Fig 1). After five days, unfed C elegans worms were almost all dead. Using E coli OP 50 as a control, viability was maintained at 74% after five days, both in larvae and in mature worm forms. By comparison, L monocytogenes lowered the viability of the worms and significantly so for the larvae forms (Mann-Whitney U test, P
